Massively-parallel cDNA sequencing has opened the way to deep and efficient probing of transcriptomes. Current approaches for transcript reconstruction from such data often rely on aligning reads to a reference genome, and are thus unsuitable for samples with a partial or missing reference genome. Here, we present the Trinity methodology for de novo full-length transcriptome reconstruction, and evaluate it on samples from fission yeast, mouse, and whitefly – an insect whose genome has not yet been sequenced. Trinity fully reconstructs a large fraction of the transcripts present in the data, also reporting alternative splice isoforms and transcripts from recently duplicated genes. In all cases, Trinity performs better than other available de novo transcriptome assembly programs, and its sensitivity is comparable to methods relying on genome alignments. Our approach provides a unified and general solution for transcriptome reconstruction in any sample, especially in the complete absence of a reference genome.

The fission yeast clade, comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus and S. japonicus, occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, suggesting a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.

The fission yeast Schizosaccharomyces pombe is a popular model system, and has been particularly influential in studies of the cell cycle and chromosome dynamics. Despite its differences from Saccharomyces cerevisiae, the tools and methods for fission yeast are conceptually similar to those used in budding yeast. Here, we present basic methods sufficient for a beginner in this system to carry out most required manipulations for genetic analysis or molecular biology.

Arrest of the cell cycle at the G2 checkpoint, induced by DNA damage, requires inhibitory phosphorylation of the kinase Cdc2 in both fission yeast and human cells. The kinase Wee1 and the phosphatase Cdc25, which regulate Cdc2 phosphorylation, were evaluated as targets of Chk1, a kinase essential for the checkpoint. Fission yeast cdc2-3w Deltacdc25 cells, which express activated Cdc2 and lack Cdc25, were responsive to Wee1 but insensitive to Chk1 and irradiation. Expression of large amounts of Chk1 produced the same phenotype as did loss of the cdc25 gene in cdc2-3w cells. Cdc25 associated with Chk1 in vivo and was phosphorylated when copurified in Chk1 complexes. These findings identify Cdc25, but not Wee1, as a target of the DNA damage checkpoint.

Patterns of replication within eukaryotic genomes correlate with gene expression, chromatin structure, and genome evolution. Recent advances in genome-scale mapping of replication kinetics have allowed these correlations to be explored in many species, cell types, and growth conditions, and these large data sets have allowed quantitative and computational analyses. One striking new correlation to emerge from these analyses is between replication timing and the three-dimensional structure of chromosomes. This correlation, which is significantly stronger than with any single histone modification or chromosome-binding protein, suggests that replication timing is controlled at the level of chromosomal domains. This conclusion dovetails with parallel work on the heterogeneity of origin firing and the competition between origins for limiting activators to suggest a model in which the stochastic probability of individual origin firing is modulated by chromosomal domain structure to produce patterns of replication. Whether these patterns have inherent biological functions or simply reflect higher-order genome structure is an open question.